Rapid industrialization in the developing countries although brought economic development but also severely degraded ecosystems of Earth such as water and soil especially by discharge coming out of the industries. This has highlighted the need for efficient processing of waste effluents discharged from the industries to prevent the degradation of ecosystems of earth. With the maturity of upcoming technologies for biofuel production for example carbon capture and sequestration technologies, gas fermentation of green-house gases, bioethanol production from lignocellulosic biomass and pyrolysis of lignocellulosic biomass, large amount of waste effluent streams are expected to be generated thus exaggerating the existing problem. Most of the effluents from biofuel industry contain large amount of organic acids, alcohols (for example formic acid, acetic acid, butyric acid, methanol and ethanol) and sugar derivatives etc. Disposing these effluents directly in environment without treatment may further impact the biodiversity of the area since these acids and alcohol are highly corrosive and toxic to living beings. Therefore these effluents must be treated chemically and biologically before discharging in environment.
Treatment of these effluent streams chemically or biologically incurs significant cost to industries thus hurting their profits however producing useful materials out of these effluents may add credit to the processes. Organic acids/alcohol particularly acetic acid are precursor of various chemicals of industrial use however extracting these from these waste effluents is commercially unsustainable due to the comparatively lower amount present in these streams and higher cost and energy footprint associated with it. These types of effluents however can be used as sources of nutrients for growth of microorganisms especially microalgae for bioproduct generation and utilization. This will not only solve the problem of processing of waste effluents but also cheap and sustainable supply of nutrient source for the cultivation of microalgae. This will add value and synergy to both the processes. However screening of microorganisms has to be done on these effluent streams since not all the microorganisms can utilize relatively higher concentration of organic acids and higher concentration of acetic acid/alcohol is reported to negatively impact growth of microbes.
Most of the natural microorganisms/microalgae are unable to tolerate or utilize higher concentration of organic acids and alcohol (particularly formic acid, acetic acid and ethanol) in the media. Therefore physiology of these microbes needs to be altered to enhance the tolerance and utilization of these nutrients in media. Genetic engineering, mutation, adaptation, protoplast fusion are such example of tools, which is being used to alter the physiology of the microbes to suit the desired application.
Most of the microalgae have ability to produce lipids in a heterotrophic mode utilizing broad range of nutrients. Heterotrophic cultivation of microalgae offers several advantages over the phototrophic cultivation like better control of culture parameters, high cell biomass and growth rate, high lipid accumulation up to 70-80% of cell dry weight in a short time. Some microalgae species such as Thraustochytrids can also produce omega-3 fatty acids i.e. DHA, DPA and EPA in significant amount along with lipids. These microorganisms can utilize broad spectrum of carbon and nitrogen source making them ideal for the economical production of both i.e. omega-3 fatty acids and biodiesel.
Most of the gas fermentation plants capturing green-house gases from different industrial sources are operated continuously instead of batch or fed batch mode. Therefore process for sequestration of nutrients from discharge of gas fermentation plants needs to be operated continuously instead of commonly used batch of fed batch process. This will not only help to synchronize gas fermentation process with nutrient sequestration process but also reduces operating cost and enhance productivity of the later process.
In continuous process, nutrients including nitrogen source are continuously supplied to the reactor, which will result into higher growth but low lipid accumulation. However oleaginous microorganisms are reported to accumulate lipid under nitrogen stress conditions. Nitrogen stress catalyses conversion of excess carbon source into lipid accumulation once nitrogen is completely consumed in the media, therefore higher carbon to nitrogen ratio is required for higher lipid production in batch or fed batch mode of cultivation. In continuous process, higher carbon to nitrogen ratio may result into higher lipid accumulation but low growth, which will translate into lower productivity of biomass, lipid and omega-3 fatty acids.
To overcome this issue, two reactors systems are applied where one reactor is meant to support higher growth and second reactor is meant for lipid accumulation under nitrogen stress conditions. However these two reactors are operated in separate batch instead of continuous mode Culture broth from first bioreactor is directly transferred to second bioreactor however this conventional approach possess numerous challenges for example (i) dilution of media components in second reactor by culture broth from first reactor, (ii) reduced biomass and lipid productivity due to drop in number of cell per ml in second reactor, (iii) chances of nitrogen contamination in second reactor caused by unutilized nitrogen present in culture broth coming from first reactor, (iv) subsequent accumulation of secondary metabolites in second reactor, which are detrimental for lipid accumulation
Based on the sources of feedstock, biodiesel has been classified into first generation, second generation, and third generation biodiesel. First generation biodiesel are being commercially produced from edible crops (e.g. Soybean Canola oil, palm oil etc.) but the viability of the first generation biodiesel in long term is however questionable because of the conflict with food supply and hence the Food vs. Fuel debate. Second generation (Non edible crops e.g. karanja oil, jatropha etc.) could be the answer for this demand but lack of adequate productivity, higher investment on water and fertilizers, vast areas of land for commercial production made limited exploiting for commercial purpose.
However, third generation biodiesel, which is derived from microbial biomass, has emerged as front runner to address the feedstock problem because of their high growth rate and oil productivity as compared to bioenergy crops. Such microorganisms belong to Yeast and Microalgae and are designated as oleaginous microorganisms, if they accumulate more than 20% of their dry weight as lipids. Oil productivity of these oleaginous microorganisms exceeds almost thousand times over superior oil crops such as palm, soybean, coconut etc and the land area needed for large scale production is far lower than oil crops (Chisti et al 2007 Biotechnology Advances). Thus cultivation of oleaginous microorganism for biodiesel application gives a commercial and sustainable edge over oil crops.
Commercial success of microalgal biodiesel depends on its cost competitiveness as compare to fossil derived fuels. The requirements for economically viable production of oil include factors such as high biomass and lipid productivity, low cost of raw material and co-product credit. A review of the microalgal biofuel indicates that cultivation of species only giving lipids for biodiesel production is not economical. The reasons include higher cultivation and nutrient cost coupled with cost associated with harvesting and oil extraction. Therefore, microalgae species which can provide value added products along with lipids are in high demand to offset some of the cost. Concurrent production of oil suited for biodiesel application and high value co-products such as DHA/DPA/EPA is one of the promising processes in order to offset the production cost thus giving leverage to biodiesel for competing against well-established petro diesel. However, selection of the good microalgae strains which can provide significant amounts of DHA/EPA/DPA remains one of the critical aspects for success of this technology.
Polyunsaturated fatty acids (PUFAs) viz. Docosahexaenoic acid (DHA), Docosapentaenoic acid (DPA) or Eicosapentaenoic acid (DPA) are the essential fatty acids and well documented for their important physiological roles in development of normal vision in infants, maintenance of brain functions, ocular tissues, heart muscles and inhibitor of macular degeneration in old people etc. Based on position of first double from end of carbon chain, PUFAs has been categorized in two class i.e. Omega-3 and omega-6 fatty acids, depend of presence of first double bond on alkyl chain opposite to carboxyl group. Broad spectrum importance of DHA in human physiology has made the companies dealing with infant food to formulate food supplement enriched with DHA, For example DHA accounts for approximately 15%-20% of lipids in the human cerebral cortex, which can be used as bio marker for analyzing the brain functioning of a particular individual, 30%-60% of lipids in the retina and is an important component of breast milk. These fatty acids are reported to have anti-inflammatory activities as well as ability to lower down blood cholesterol level. Regular dozes of DHA can be helpful to prevent the atherosclerosis by reducing the risk of blood vessel hardening. Recently it was reported that long term use of DHA can be helpful to reduce the chance of type-1 diabetes, non-alcoholic fat liver diseases and cancer by inducing apoptosis in cancerous cells. DHA application in cosmetics is reported to have anti-aging effect. Because omega-3 fatty acids are not synthesized de novo in the human body, these fatty acids must be derived from nutritional sources.
The broad spectrum of physiological importance, DHA/EPA is drawing significant attention from pharmaceutical industry, neutraceutical industry, poultry, and fisheries. Recently attempts have been made to expand DHA application in functional and health foods. World Health Organization (WHO) has suggested the daily intake of 1 g/day DHA for healthy persons. Due to their expanding application in various industries, market value for these omega-3 products is projected to be around US $35 billion by 2016 with compound annual growth rate of 6.8% from 2011-2016 (Global market for EPA/DHA Omega-3 Products, Packaged Facts 2012).
Fish oil is considered good dietary sources of omega-3 fatty acids. Fish oils vary considerably in the type and level of fatty acid composition depending on the particular species and their diets. For example, fish raised by aquaculture tend to have a lower level of omega-3 fatty acids than those in the wild. Furthermore, fish oils carry the risk of containing environmental contaminants and can be associated with stability problems and a fishy odor or taste. Apart from this, Overfishing has also led to the faltering fish oil supply which forced the major producers such as DSM to increase the market price for DHA due to cost escalation in fish oil procurement.
These limiting issues with fish oil supply have forced the industries to look for alternative resources such as marine microalgae like Thraustochytrids, Crypthecodinum, or diatoms Phaeodactylum for DHA/EPA production in last two decades. Significant amount of research has been conducted for isolation of omega-3 oil producing marine microalgae. Thraustochytrids are deemed as one of the potential candidates for DHA production. Thraustochytrids are unicellular marine micro-heterotroph, associated with the degrading organic materials such as mangroves leaves, sediment and recycling of nutrients in mangrove ecosystem. Cell size ranges between 10-35 μm and reproduce through zoospores or binary fission. They act as decomposer in mangrove ecosystem. Based on molecular phylogeny, Thraustochytrids are classified in the Kingdom Chromista or Stramenipila (also called Stramenopila), alongside brown algae, diatoms, oomycetes and a variety of flagellates. While most of the members of this kingdom are saprophytic such as Thraustochytrids, some are the parasitic like member of labyrinthulids. High Biomass and Lipid production including DHA/EPA is well known to be produced by microalgae belongs to Thraustochytrids in nature. Thraustochytrids are microorganisms of the order Thraustochytriales and include members of the genus Schizochytrium, Thraustochytrium, Ulkenia and have been well established as an alternative source of omega-3 fatty acids, including DHA
Out of total lipid present in thraustochytrid biomass, about 90%-95% is stored as triacyl glycerols (TAGs) followed by 5%-10% as polar and structural lipids (Gupta et al 2012 Biotechnology Advances). Saturated fatty acids (SFAs) such as Myristic acid (C14:0), Palmitic acid (C16:0) or mono unsaturated fatty acids e.g. Oleic acid (C18:1) and polyunsaturated fatty acids (PUFAs) like DHA (C22:6n3) are the major component of TAGs. SFAs and MUFAs constitute about 40%-80% of total fatty acids (TFAs) which are ideal fatty acids for biodiesel production (FABs). DHA constitutes 20%-50% of TFA. In last two decades, most of the research done for the development of Thraustochytrids is for development of a novel source of DHA production. Literature suggests that high biomass production (200 g/L) with high lipid accumulation (SFAs and MUFAs) makes them potential candidate for the concurrent production of biodiesel and high value co-products.
Thraustochytrids are well documented for their ability to produce high amount of biomass and lipid coupled with DHA/EPA production using variety of carbon and nitrogen sources. However source of carbon and nitrogen supply whose costs also impacts the overall production economics has to be carefully chosen. Glycerol, crude glycerol, coconut water, soybean meal and sweet sorghum juice are few examples of cheap nutrients. Glycerol especially crude glycerol from biodiesel industry has been aggressively advocated as alternative of glucose for the Thraustochytrid cultivation by researchers. However with the shifting of focus of biodiesel industry from non-edible oil to microalgal oil as feedstock of biodiesel, supply and cost dynamics of crude glycerol would also change resulting into drop in carbon source supply for Thraustochytrid cultivation. It has been observed that the cost of the carbon source is a single most significant factor for overall economics of lipid/DHA production.
Thraustochytrids are exclusively marine microbes, cultivated in media having sea water or sea salt. There are some reports suggesting the replacement of sea water or sea salt with sodium chloride or sodium sulphate. However addition of sea salt, sea water, sodium chloride and sodium sulphate are reported to corrode the walls of commercial scale reactors in long term operations. Thus media formulations without the inclusion of sea salt, sea water, sodium chloride and sodium sulphate will have multiple advantages for example easier maintenance of reactors, reduced likelihoods of corrosive metal contamination in media, monetary benefits in maintenance of reactors and media preparations.
Patent publication number US 2013/0065282 describes the process of anaerobic fermentation of green-house gases such as carbon dioxide along with hydrogen into ethanol and acids. This stream was later used as carbon source for the production of lipids by oleaginous yeasts for example Cryptococcus curvatus. It is disclosed in this that 1.5% w/v acetate was used in the media for the cultivation since use of more than 1 to 1.5% of acetate is reported to negatively affect the growth. Therefore waste effluent streams containing more than 1.5% of acetate may restrict the growth of these oleaginous yeasts. Author claimed lipid productivity of 20 g/L/d but lipid content remained very low in dry biomass of the said yeast. Dry biomass of this yeast also lack of any highly valuable product which may discourage the industry to employ this platform for processing of waste effluents.
US patent application number US 2012/0198758 A1 discusses the conversion of municipal sewage stream into bio crude oil by employing hydrothermal process on wet algal biomass and biosolid fraction of municipal waste. The used stream was mentioned to be rich in human waste, food waste, and pharmaceutical waste or used water supply of community. The process involved heating of solid fraction, wet biomass of algae and bacteria at very high temperature and pressure for the conversion into biocrude oil. Although this process eliminated the need of biomass drying, selection of high oil content microorganisms but energy required to carry out hydrothermal process was higher than the energy content of the biocrude oil. Apart from that any high value coproduct extractable from algal biomass will be also denatured at such harsh conditions of hydrothermal reactor, thus reducing the possibility of coproduct credit during the conversion of municipal waste to biocrude oil, which will in turn dampen the industries to adopt such technologies for processing their waste effluents. However processes converting these waste effluents into biodiesel and high value products such as DHA will be of significant interest.
U.S. Pat. No. 5,130,242 (1992) describes the process for heterotrophic cultivation of microalgae which can be used for extracting omega-3 fatty acids. Claimants of the patent describes about the isolation of a fast growing Thraustochytrids and its subsequent cultivation on glucose or other sugars such as maltose, sucrose or starch for enhancing DHA content in the cell. A two stage fermentation strategy was employed in this patent, where first stage was exponential growth phase followed by lipid accumulation phase under nitrogen stress conditions. Authors also described the process of low temperature crystallization for separation and purification of omega-3 fatty acids from rest of the lipid and effect of sodium ion concentration on fatty acid accumulation. The whole cell extract of Thraustochytrids was later suggested to be used in the foods for nutritional supplement or fish or animal feed to enhance omega-3 content in the product derived from these industries for example fish oil, eggs etc. However, use of glucose or other commoditized carbon and nitrogen sources will not only escalate the production cost but also poses challenging question for long term supply of these commodities along with raising the commodity prices.
U.S. Pat. No. 6,582,941 B1 (2003) describes the isolation of fast growing thraustochytrid strains belonging to the species similar to Schizochytrium limacinum SR21. Claimants studied this strain to develop the process for producing lipid having omega-3 and omega-6 fatty acids in substantial percentage using glucose or glycerol as carbon source and ammonium salts along with corn steep liquor as nitrogen sources. Later they optimized the growth of this newly isolated strain Schizochytrium limacinum SR21 to enhance omega-3 and omega-6 fatty acids content. Different media formulations of carbon and nitrogen sources were tried to increase DHA production in the fermentation. However the choice of carbon source as glucose or glycerol may makes this process rather uneconomical and non-sustainable.
U.S. Pat. No. 6,607,900 mentions the process for high cell density cultivation of Schizochytrium strain with biomass from 17 g/L to 200 g/L with significant amount of lipid accumulation using glucose as carbon source. Patent claimed DHA content around 20%-25% w/w of total lipid. Stepwise aeration strategy was applied in this patent with high aeration rate at growth phase followed by low aeration rate at lipid accumulation phase once the nitrogen source i.e. ammonium hydroxide is totally consumed in the medium.
US 20090117194 describes the process for concurrent production of DHA with antioxidant production with novel thraustochytrid strain ONC-T18, which claimants isolated from Canadian marine biodiversity. 18S rDNA gene sequence of this isolate revealed its proximity with Thraustochytrium striatum T91-6. They used different concentration of glucose, monosodium glutamate and yeast extract to enhance biomass and DHA content in the cell. After trying several media formulations they reported substantial increase in biomass and lipid content up to 28.0 g L-1 biomass, 81.7% TFA and 31.4% DHA (w/w biomass).
U.S. Pat. No. 7,989,195 describes the process to achieve high cell density cultivation of Schizochytrium limacinum SR21 using crude glycerol as carbon source. Claimants present multiphase strategy dividing the whole fermentation in three phase i.e. increasing cell density, cell size and subsequent increase in fatty acid production. They claimed biomass productivity of 1 g/L/h to 3 g/L/h with 15%-22% DHA of total fatty acids. The crude glycerol used in this process was derived from biodiesel industry. Authors did not mention the effect of shearing on lipid accumulation since obese cells are very delicate and prone to cell damage due to shearing thus effect of shearing has to be taken into account while enhancing the lipid accumulation in the cell.
A US Patent application number 2013/0217084 A1 describes a process for DHA production from various thraustochytrid species i.e. Schizochytrium, Thraustochytrium, Ulkenia etc. using crude glycerol, generated from biodiesel industry, as carbon source. Claimants also studied EPA production in these species on crude glycerol. They mentioned about the value addition to biodiesel industry by converting acetate containing glycerol in to high value products.
Patent publication number US 2013/0089901 describes that the microalgae of the invention accumulate bio-oil at a high ratio in the cells when being cultured in glucose-containing medium, and thus can produce bio-oil in a high yield. The microalgae can produce bio-oil using lignocellulosic biomass as a carbon source. Moreover, the use of cellulosic biomass for production of bio-oil can overcome the factors limiting the development of bio-oil, including the unstable supply of food resources and an increase in the cost of raw materials, and can improve the commercial competitiveness of microbial fermentation oil. However, the effect of presence of toxic compounds in the pretreated biomass which are known to inhibitory to most of organisms was not studied.
WO 2007/068997 mentions the isolation and characterization of unreported 10 thraustochytrid strains from Goa, India for DHA production. Claimants screened these isolates on 2% glucose as carbon source to assess their ability for DHA accumulation and other related intermediate fatty acids. Authors claimed 10%-30% DHA content of total fatty acids in some of the isolates, however use of glucose as carbon source for DHA production raises serious question on the production cost along with long term supply of glucose for industrial scale DHA production when supply of cheap sources of glucose are already under pressure.
Patent publication number US2010/0041112 presents the application of lipid extracted from Thraustochytrid strains for biodiesel production. The strains used in this invention were extremely rich in SFA or MUFA. Use of glucose and yeast extract in the medium will translate into substantial escalation in production cost thus endangering the commercial viability of the process and long term sustainability both.
Addition of sea salt in the media is necessary for the Thraustochytrid cultivation however this may cause corrosion in large scale steal reactors and associated hardware thus to avoid this sea salt is replaced with non-chloride sodium salts such as sodium sulphate (US Pat. No. 5,340,742). However addition of non-chloride sodium salts in waste effluent stream will add further cost to the processing of waste streams. Therefore a process devoid of sea salt or sodium salt will be really helpful to integrate this process for processing of waste effluents
From the prior art, it can concluded that use of other carbon sources other than glucose also poses the same question since long term supply of these carbon sources for large scale cultivation is questionable, if one is aiming for to use them as feedstock for biodiesel production. This will increase the carbon foot print of the process as well; an issue global community is grappling with. However integrating waste effluents processing with Thraustochytrid cultivation will add value to both the processes of waste effluents processing and biodiesel, DHA production from Thraustochytrid cultivation. This will in turn increase the commercial viability and sustainability of the process along with reducing carbon foot print of the process.